The soybean (Glycine max) belongs to the Fabaceae (Leguminosae) family. The soybean is thought to have originated in China. Wild types of soybeans are viny in nature, which may explain why soybeans were first introduced in the United States as a hay crop. Introductions from China, Manchuria, Korea and Japan have been important in developing varieties for the United States. Modern breeding efforts to improve the agronomic traits, such as more erect growth, reduced lodging and increased seed size, have been primarily responsible for the development of soybeans into a crop of worldwide importance. The acreage and the proportion of the crop harvested for grain has increased steadily and today soybeans are a major world commodity.
Cultivated soybean has a substantial commercial value throughout the world. Over 50 million hectares worldwide are used to produce an annual crop of soybeans in excess of 100 metric tons with an estimated value exceeding 20 billion dollars. The development of scientific methods useful in improving the quantity and quality of this crop is, therefore, of significant commercial interest. Soybeans are widely used as a source of protein, oil, condiments and chemical feedstock. Significant effort has been expended to improve the quality of cultivated soybean species by conventional plant breeding, and a number of major successes are recorded. The methods of conventional plant breeding have been limited, however, to the movement of genes and traits from one soybean variety to the other.
Modern biotechnological research and development has provided useful techniques for the improvement of agricultural products by plant genetic engineering. Plant genetic engineering involves the transfer of a desired gene or genes into the inheritable germline of crop plants such that those genes can be bred into or among the elite varieties used in modern agriculture. Gene transfer techniques allow the development of new classes of elite crop varieties with improved disease resistance, herbicide tolerance, and increased nutritional value. Various methods have been developed for transferring genes into plant tissues including high velocity microprojection, microinjection, electroporation, direct DNA uptake, and Agrobacterium-mediated gene transformation. Although widely used for dicotyledonous plants, DNA delivery using particle bombardment, electroporation, or Agrobacterium-mediated delivery into soybean has proven to be difficult. This is due, in part, to the small number of cells that have been found to be totipotent in soybean (Trick 1997). Two methods routinely used are an Agrobacterium-based method targeting the cotyledonary-node axillary meristems (Hinchee 1988) and a method using particle bombardment of mature zygotic embryos (Finer 1991).
The lack of effective selective agents is one of the bottlenecks in the efficiency of different soybean transformation methods. The efficacy of tissue culture selection systems depends on many factors including tissue type, size of explant, chemical characteristics of the selectable agent and concentrations and time of application. The most used method of selection is known as negative selection, which employs selection markers that confer resistance against a phytotoxic agent (such as an herbicide or antibiotic). The negative selection markers employed so far are mainly limited to neomycin 3′-O-phosphotransferase (nptII), phosphinothricin acetyltransferases (PAT; also named Bialophos® resistance; bar; de Block 1987; EP 0 333 033; U.S. Pat. No. 4,975,374), 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; conferring resistance to Glyphosate® (N-(phosphonomethyl)glycine); and hygromycin B. Alternative selection marker systems, such as a system based on D-amino acid metabolizing enzymes (e.g., D-amino acid dehydratases or oxidases), has been recently described on a general basis (WO 03/060133; Erikson 2004). However, no adoption and/or optimization of such a system for use in soybean has been described so far. Accordingly, the object of the present invention is to provide an improved, efficient method for transforming Glycine max plants based on D-amino acid selection. This objective is achieved by the present invention.
Although some of the problems linked to the transformation of soybeans have been overcome by the methods described in the art, there is still a significant need for improvement, since all methods known so far have only a low to moderate transformation and—especially—regeneration efficiency. Although significant advances have been made in the field of Agrobacterium-mediated transformation methods, a need continues to exist for improved methods to facilitate the ease, speed and efficiency of such methods for transformation of soybean plants. Therefore, it was the objective of the present invention to provide an improved method having higher overall efficiency in the process of generation of transgenic soybean plants. This objective is solved by the present invention.